It has been shown previously that subendocardial (ENDO) and subepicardial (EPI) cells of ventricular wall differ in their mechanical properties. The subendocardial myocyte displayed a longer time to peak contraction and delayed relaxation in different animal species (1, 2). Although cardiomyocytes are exposed to mechanical load in ‘in situ’ beating heart, these studies were performed using mechanically unloaded cells. The aim of our study was to investigate the differences in the responses to stretch (mechanical preload) between EPI and ENDO cells from mouse left ventricle (LV) during auxotonic contractions. All studies were conducted according to UK legislation. EPI and ENDO ventricular myocytes were enzymatically isolated from hearts excised from C57BL/6 mice (aged 9-11 weeks) and stored in normal Tyrode solution. Each cell end was hold by a pair of carbon fibers to apply 3-5 % axial stretch to the cells (3). Cells were stimulated at 1 Hz at room temperature. All values are presented as means ± SEM. Student’s unpaired t-test and two-way ANOVA were used for statistical analysis. To predict electromechanical mechanisms responsible for the differences, we utilized our mathematical EPI and ENDO cell models that describe transmural gradient between the cells in some ionic currents and myofilament contractile mechanisms (4). Although there were found significant differences neither in stiffness (slope of end-diastolic force-length relationship, EDSFLR) nor in contractility (slope of end-systolic force-length relationship, ESFLR) between ENDO and EPI cells, ENDO cells tended to show steeper slope in ESFLR (ENDO: 0.43±0.08 nN/mm3, n = 28, vs EPI: 0.40±0.07, n = 22). ENDO cells showed significantly longer time to peak contraction (Tmax) compared to EPI cells during auxotonic contractions at non-stretched state (ENDO: 125.3±5.0 ms, n = 26, vs EPI: 110.6±3.5, n = 20, p<0.05). We did not find significant differences in the time constant of relaxation (τ) (ENDO: 39.46±1.81 ms, n = 26, vs EPI: 39.55±2.32, n = 20) between the groups. Stretch delayed Tmax in both groups while EPI cells showed significantly greater delay in the Tmax compared with ENDO cells, resulting a smaller difference in Tmax at stretched state (ENDO: 129.8±5.6 ms vs EPI: 118.8±3.9). Modeling results suggest that differences in the kinetics of cross bridges and calcium-troponin C complexes assumed in ENDO and EPI models may essentially contribute to the differences in the load-dependency between the cells. The present results demonstrate that transmural gradient in the characteristics of cellular contractile profile decreases under stretch that may lead to synchronization of contractions of LV in the intact heart.